Interest in leaf protein concentrates for animal and human consumption has increased in recent years as a result of an anticipated world-wide need for alternate protein sources and a desire to derive maximum usefulness from agricultural crops. In order to develop alternate methods for purification of leaf proteins as potential nutritional supplements, I have found methods to purify and crystallize fraction I protein from a number of plant species, particularly crystallization of the fraction I protein of plants which are major agricultural crops and have not been previously obtained. The methods of the present invention apply to any photosynthetic organisms.
Ribulose-1,5-bisphosphate carboxylase/oxygenase [E.C.,4.1.1.39], fraction I protein, is a complex, multimeric enzyme found in a variety of photosynthetic organisms which possesses two apparently counterproductive activities. Ribulose-1,5-biphosphate carboxylase/oxygenase reacts with CO.sub.2 in an initial reaction of the well known C.sub.3 photosynthetic carbon reduction cycle and catalyzes a carboxylation reaction of ribulose-1,5-bisphosphate to yield two moles of 3-D-phosphoglycerate. Molecular oxygen, if present, will compete with CO.sub.2 for the catalytic site, resulting in the oxygenation of ribulose-1,5-bisphosphate. The products of this initial reaction in the C.sub.2 photorespiratory carbon oxidation cycle are 3-D-phosphoglycerate and 2-phosphoglycolate. Net flux of glycolate from the plant chloroplast provides the substrate for photorespiration which results in a net loss of fixed carbon as CO.sub.2.
The ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme from eukaryotic sources sediments at 18S (Svedberg constant), has a molecular weight of approximately 550,000 and is composed of eight each of large and small subunits. The large subunits, which contain the catalytically-active site, have a molecular weight of approximately 55,000 and are coded by chloroplast DNA. In contrast, the genetic information for the small subunits (15,000 molecular weight) is located in the nuclear genome. Although believed to be regulatory in nature, the precise function of the small subunits remains enigmatic.
The forces which cause ribulose-1,5-bisphosphate carboxylase/oxygenase, and other proteins, to crystallize are complex and poorly understood. Solubility properties of proteins differ widely and are a function of the protein under investigation. Therefore, to develop a successful crystallization strategy, one must identify and understand the solubility properties of the particular protein of interest. A precipitant can be used to aid in bringing about supersaturation in order to induce nucleation and crystallization of macromolecules. Particular attention has been placed on selecting an appropriate precipitant and determining the concentrations required for crystallization. Polyethylene glycol 4000, 6000 and (NH.sub.4).sub.2 SO.sub.4 have been found to be effective precipitants, under controlled pH conditions disclosed herein, for crystallization of ribulose-1,5-biphosphate carboxylase/oxygenase from many diverse plant species. The crystallization methods described herein may vary slightly between various plant species and guidelines are provided for crystallization of fraction I proteins from leaves of alfalfa, tomato, corn, spinach, cotton, potato and tobacco to enable crystallization from essentially any photosynthetic organisms.